Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA
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Curated by eLife
Evaluation Summary:
This manuscript is a timely contribution to the CRISPR/Cas field: the mode of function of the type III-E Cas7-11 CRISPR-Cas system. This is an RNA-guided RNA targeting system only characterized last year. In contrast to Cas13 systems, Cas7-11 does not possess collateral damaging activity, hence does not show cytotoxicity when introduced into human cells. These are highly desirable traits in practical applications. High resolution mechanistic studies would be essential for driving the further development of Cas7-11 based biotechnology applications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.
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Author Response
Reviewer #1 (Public Review):
High resolution mechanistic studies would be instrumental in driving the development of Cas7-11 based biotechnology applications. This work is unfortunately overshadowed by a recent Cell publication (PMID: 35643083) describing the same Cas7-11 RNA-protein complex. However, given the tremendous interest in these systems, it is my opinion that this independent study will still be well cited, if presented well. The authors obviously have been trying to establish a unique angle for their story, by probing deeper into the mechanism of crRNA processing and target RNA cleavage. The study is carried out rigorously. The current version of the manuscript appears to have been rushed out. It would benefit from clarification and text polishing.
We thank the reviewer for the positive and helpful …
Author Response
Reviewer #1 (Public Review):
High resolution mechanistic studies would be instrumental in driving the development of Cas7-11 based biotechnology applications. This work is unfortunately overshadowed by a recent Cell publication (PMID: 35643083) describing the same Cas7-11 RNA-protein complex. However, given the tremendous interest in these systems, it is my opinion that this independent study will still be well cited, if presented well. The authors obviously have been trying to establish a unique angle for their story, by probing deeper into the mechanism of crRNA processing and target RNA cleavage. The study is carried out rigorously. The current version of the manuscript appears to have been rushed out. It would benefit from clarification and text polishing.
We thank the reviewer for the positive and helpful comments that have made the manuscript more impactful.
To summarize the revisions, we have resolved the metal-dependence issue, updated the maps in both main and supplementary figures that support the model, re-organized the labels for clarity, and added the comparison between our and Kato et al.’ structures.
In addition, we describe a new result with an isolated C7L.1 fragment that retains the processing and crRNA binding activities.
Reviewer #2 (Public Review):
In this manuscript, Gowswami et al. solved a cryo-EM structure of Desulfonema ishimotonii Cas7-11 (DiCas7-11) bound to a guiding CRISPR RNA (crRNA) and target RNA. Cas7-11 is of interest due to its unusual architecture as a single polypeptide, in contrast to other type III CRISPR-Cas effectors that are composed of several different protein subunits. The authors have obtained a high-quality cryo-EM map at 2.82 angstrom resolution, allowing them to build a structural model for the protein, crRNA and target RNA. The authors used the structure to clearly identify a catalytic histidine residue in the Cas7-11 Cas7.1 domain that is important for crRNA processing activity. The authors also investigated the effects of metal ions and crRNAtarget base pairing on target RNA cleavage. Finally, the authors used their structure to guide engineering of a compact version of Cas7-11 in which an insertion domain that is disordered in the cryo-EM map was removed. This compact Cas7-11 appears to have comparable cleavage activity to the full-length protein.
The cryo-EM map presented in this manuscript is generally of high quality and the manuscript is very well illustrated. However, some of the map interpretation requires clarification (outlined below). This structure will be valuable as there is significant interest in DiCas7-11 for biotechnology. Indeed, the authors have begun to engineer the protein based on observations from the structure. Although characterization of this engineered Cas7-11 is limited in this study and similar engineering was also performed in a recently published paper (PMID 35643083), this proof-of-principle experiment demonstrates the importance of having such structural information.
The biochemistry experiments presented in the study identify an important residue for crRNA processing, and suggest that target RNA cleavage is not fully metal-ion dependent. Most of these conclusions are based on straightforward structure-function experiments. However, some results related to target RNA cleavage are difficult to interpret as presented. Overall, while the cryo-EM data presented in this work is of high quality, both the structural model and the biochemical results require further clarification as outlined below.
We thank the reviewer for the positive and helpful comments that have made the manuscript more impactful.
To summarize the revisions, we have resolved the metal-dependence issue, updated the maps in both main and supplementary figures that support the model, re-organized the labels for clarity, and added the comparison between our and Kato et al.’ structures.
In addition, we describe a new result with an isolated C7L.1 fragment that retains the processing and crRNA binding activities.
- The DiCas7-11 structure bound to target RNA was also recently reported by Kato et al. (PMID 35643083). The authors have not cited this work or compared the two structures. While the structures are likely quite similar, it is notable that the structure reported in the current paper is for the wild-type protein and the sample was prepared under reactive conditions, resulting in a partially cleaved target. Kato et al. used a catalytically dead version of Cas7-11 in which the target RNA should remain fully intact. Are there differences in the Cas7-11 structure observed in the presence of a partially cleaved target RNA in comparison to the Kato et al. structure? Such a comparison is appropriate given the similarities between the two reports. A figure comparing the two structures could be included in the manuscript.
We have added a paragraph on page 12 that describe the differences in preparation of the two complexes and their structures. We observed minor differences in the overall protein structure (r.m.s.d. 0.918 Å for 8114 atoms) but did observe quite different interactions between the protein and the first 5’-tag nucleotide (U(-15) vs. G(-15)) due to the different constructs in pre-crRNA, which suggests an importance of U(-15) in forming the processing-competent active site. We added Figure 2-figure supplementary 3 that illustrates the similarities and the differences.
2.The cryo-EM density map is of high quality, but some of the structural model is not fully supported by the experimental data (e.g. protein loops from the alphafold model were not removed despite lack of cryo-EM density). Most importantly, there is little density for the target RNA beyond the site 1 cleavage site, suggesting that the RNA was cleaved and the product was released. However, this region of the RNA was included in the structural model. It is unclear what density this region of the target RNA model was based on. Further discussion of the interpretation of the partially cleaved target RNA is necessary. Were 3D classes observed in various states of RNA cleavage and with varied density for the product RNAs?
We should have made it clear in the Method that multiple maps were used in building the structure but only submitted the post-processed map to reviewers. When using the Relion 4.0’s local resolution estimation-generated map, we observed sufficient density for some of the regions the reviewer is referring to. For instance, the site 1 cleavage density does support the model for the two nucleotides beyond site 1 cleavage site (see the revised Figure 1 & Figure 1- figure supplement 3).
However, there are protein loops that remain lack of convincing density. These include 134141 and 1316-1329 that are now removed from the final coordinate.
The “partially cleaved target RNA” phrase is a result of weak density for nucleotides downstream of site 1 (+2* and +3) but clear density flanking site 2. This feature indicates that cleavage likely had taken place at site 1 but not site 2 in most of the particles went into the reconstruction. To further clarify this phrase, we added “The PFS region plus the first base paired nucleotide (+1) are not observed.” on page 4 and better indicate which nucleotides are or are not built in our model in Figure 1.
- The authors argue that site 1 cleavage of target RNA is independent of metal ions. This is a potentially interesting result, but it is difficult to determine whether it is supported by the evidence provided in the manuscript. The Methods section only describes a buffer containing 10 mM MgCl2, but does not describe conditions containing EDTA. How much EDTA was added and was MgCl2 omitted from these samples? In addition, it is unclear whether the site 1 product is visible in Figures 2d and 3d. To my eye, the products that are present in the EDTA conditions on these gels migrated slightly slower than the typical site 1 product. This may suggest an alternate cleavage site or chemistry (e.g. cyclic phosphate is maintained following cleavage). Further experimental details and potentially additional experiments are required to fully support the conclusion that site 1 cleavage may be metal independent.
As we pointed out in response to Reviewer 1’s #8 comment, this conclusion may have been a result of using an older batch of DiCas7-11 that contains degraded fragments.
As shown in the attached figure below, “batch Y” was an older prep from our in-house clone and “batch X” is a newer prep from the Addgene purchased clone (gel on right), and they consistently produce metal-independent (batch Y) or metal-dependent (batch X) cleavage (gel on left). It is possible that the degraded fragments in batch Y carry a metal-independent cleavage activity that is absent in the more pure batch X.
We further performed mass spectrometry analysis of two of the degraded fragments from batch Y (indicated by arrows below) and discovered that these are indeed part of DiCas7-11. We, however, cannot rationalize, without more experimental evidence, why these fragments might have generated metal-independent cleavage at site 1. Therefore, we simply updated all our cleavage results from the new and cleaner prep (batch X) (For instance, Figure 3c). As a result, all references to “metal-independence” were removed.
With regard to the nature of cleaved products, we found both sites could be inhibited by specific 2’-deoxy modifications, consistent with the previous observation that Type III systems generate a 2’, 3’-cyclic product in spite of the metal dependence (for instance, see Hale, C. R., Zhao, P., Olson, S., Duff, M. O., Graveley, B. R., Wells, L., ... & Terns, M. P. (2009). RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell, 139(5), 945-956.)
We added this rationale based on the new results and believe that these characterizations are now thorough and conclusive
- The authors performed an experiment investigating the importance of crRNA-target base pairing on cleavage activity (Figure 3e). However, negative controls for the RNA targets in the absence of crRNA and Cas7-11 were not included in this experiment, making it impossible to determine which bands on the gel correspond to substrates and which correspond to products. This result is therefore not interpretable by the reader and does not support the conclusions drawn by the authors.
Our original gel image (below) does contain these controls but we did not include them for the figure due to space considerations (we should have included it as a supplementary figure). We have now completely updated Figure 3e with much better quality and controls. Both the older and the updated experiments show the same results.
Original gel for Figure 3e containing controls.
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Evaluation Summary:
This manuscript is a timely contribution to the CRISPR/Cas field: the mode of function of the type III-E Cas7-11 CRISPR-Cas system. This is an RNA-guided RNA targeting system only characterized last year. In contrast to Cas13 systems, Cas7-11 does not possess collateral damaging activity, hence does not show cytotoxicity when introduced into human cells. These are highly desirable traits in practical applications. High resolution mechanistic studies would be essential for driving the further development of Cas7-11 based biotechnology applications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
High resolution mechanistic studies would be instrumental in driving the development of Cas7-11 based biotechnology applications. This work is unfortunately overshadowed by a recent Cell publication (PMID: 35643083) describing the same Cas7-11 RNA-protein complex. However, given the tremendous interest in these systems, it is my opinion that this independent study will still be well cited, if presented well. The authors obviously have been trying to establish a unique angle for their story, by probing deeper into the mechanism of crRNA processing and target RNA cleavage. The study is carried out rigorously. The current version of the manuscript appears to have been rushed out. It would benefit from clarification and text polishing.
-
Reviewer #2 (Public Review):
In this manuscript, Gowswami et al. solved a cryo-EM structure of Desulfonema ishimotonii Cas7-11 (DiCas7-11) bound to a guiding CRISPR RNA (crRNA) and target RNA. Cas7-11 is of interest due to its unusual architecture as a single polypeptide, in contrast to other type III CRISPR-Cas effectors that are composed of several different protein subunits. The authors have obtained a high-quality cryo-EM map at 2.82 angstrom resolution, allowing them to build a structural model for the protein, crRNA and target RNA. The authors used the structure to clearly identify a catalytic histidine residue in the Cas7-11 Cas7.1 domain that is important for crRNA processing activity. The authors also investigated the effects of metal ions and crRNA-target base pairing on target RNA cleavage. Finally, the authors used their …
Reviewer #2 (Public Review):
In this manuscript, Gowswami et al. solved a cryo-EM structure of Desulfonema ishimotonii Cas7-11 (DiCas7-11) bound to a guiding CRISPR RNA (crRNA) and target RNA. Cas7-11 is of interest due to its unusual architecture as a single polypeptide, in contrast to other type III CRISPR-Cas effectors that are composed of several different protein subunits. The authors have obtained a high-quality cryo-EM map at 2.82 angstrom resolution, allowing them to build a structural model for the protein, crRNA and target RNA. The authors used the structure to clearly identify a catalytic histidine residue in the Cas7-11 Cas7.1 domain that is important for crRNA processing activity. The authors also investigated the effects of metal ions and crRNA-target base pairing on target RNA cleavage. Finally, the authors used their structure to guide engineering of a compact version of Cas7-11 in which an insertion domain that is disordered in the cryo-EM map was removed. This compact Cas7-11 appears to have comparable cleavage activity to the full-length protein.
The cryo-EM map presented in this manuscript is generally of high quality and the manuscript is very well illustrated. However, some of the map interpretation requires clarification (outlined below). This structure will be valuable as there is significant interest in DiCas7-11 for biotechnology. Indeed, the authors have begun to engineer the protein based on observations from the structure. Although characterization of this engineered Cas7-11 is limited in this study and similar engineering was also performed in a recently published paper (PMID 35643083), this proof-of-principle experiment demonstrates the importance of having such structural information.
The biochemistry experiments presented in the study identify an important residue for crRNA processing, and suggest that target RNA cleavage is not fully metal-ion dependent. Most of these conclusions are based on straightforward structure-function experiments. However, some results related to target RNA cleavage are difficult to interpret as presented. Overall, while the cryo-EM data presented in this work is of high quality, both the structural model and the biochemical results require further clarification as outlined below.
1. The DiCas7-11 structure bound to target RNA was also recently reported by Kato et al. (PMID 35643083). The authors have not cited this work or compared the two structures. While the structures are likely quite similar, it is notable that the structure reported in the current paper is for the wild-type protein and the sample was prepared under reactive conditions, resulting in a partially cleaved target. Kato et al. used a catalytically dead version of Cas7-11 in which the target RNA should remain fully intact. Are there differences in the Cas7-11 structure observed in the presence of a partially cleaved target RNA in comparison to the Kato et al. structure? Such a comparison is appropriate given the similarities between the two reports. A figure comparing the two structures could be included in the manuscript.
2. The cryo-EM density map is of high quality, but some of the structural model is not fully supported by the experimental data (e.g. protein loops from the alphafold model were not removed despite lack of cryo-EM density). Most importantly, there is little density for the target RNA beyond the site 1 cleavage site, suggesting that the RNA was cleaved and the product was released. However, this region of the RNA was included in the structural model. It is unclear what density this region of the target RNA model was based on. Further discussion of the interpretation of the partially cleaved target RNA is necessary. Were 3D classes observed in various states of RNA cleavage and with varied density for the product RNAs?
3. The authors argue that site 1 cleavage of target RNA is independent of metal ions. This is a potentially interesting result, but it is difficult to determine whether it is supported by the evidence provided in the manuscript. The Methods section only describes a buffer containing 10 mM MgCl2, but does not describe conditions containing EDTA. How much EDTA was added and was MgCl2 omitted from these samples? In addition, it is unclear whether the site 1 product is visible in Figures 2d and 3d. To my eye, the products that are present in the EDTA conditions on these gels migrated slightly slower than the typical site 1 product. This may suggest an alternate cleavage site or chemistry (e.g. cyclic phosphate is maintained following cleavage). Further experimental details and potentially additional experiments are required to fully support the conclusion that site 1 cleavage may be metal independent.
4. The authors performed an experiment investigating the importance of crRNA-target base pairing on cleavage activity (Figure 3e). However, negative controls for the RNA targets in the absence of crRNA and Cas7-11 were not included in this experiment, making it impossible to determine which bands on the gel correspond to substrates and which correspond to products. This result is therefore not interpretable by the reader and does not support the conclusions drawn by the authors.
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